+

WO2008010986A2 - Procédés de modulation de l'angiogenèse - Google Patents

Procédés de modulation de l'angiogenèse Download PDF

Info

Publication number
WO2008010986A2
WO2008010986A2 PCT/US2007/016127 US2007016127W WO2008010986A2 WO 2008010986 A2 WO2008010986 A2 WO 2008010986A2 US 2007016127 W US2007016127 W US 2007016127W WO 2008010986 A2 WO2008010986 A2 WO 2008010986A2
Authority
WO
WIPO (PCT)
Prior art keywords
egfl7
angiogenesis
agonist
administering
tumor
Prior art date
Application number
PCT/US2007/016127
Other languages
English (en)
Other versions
WO2008010986A3 (fr
Inventor
Calvin Jay Kuo
Frank Kuhnert
Original Assignee
The Board Of Trustees Of The Leland Stanford Junior University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Board Of Trustees Of The Leland Stanford Junior University filed Critical The Board Of Trustees Of The Leland Stanford Junior University
Publication of WO2008010986A2 publication Critical patent/WO2008010986A2/fr
Publication of WO2008010986A3 publication Critical patent/WO2008010986A3/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • Angiogenesis and vasculogenesis are processes involved in the growth of blood vessels.
  • Angiogenesis is the process by which new blood vessels are formed from extant capillaries, while vasculogenesis involves the growth of vessels deriving from endothelial progenitor cells.
  • Angiogenesis is a complex, combinatorial process that is regulated by a balance between pro- and anti-angiogenic molecules.
  • Angiogenic stimuli e.g. hypoxia or inflammatory cytokines
  • VEGF vascular endothelial growth factor
  • FGF fibroblast growth factor
  • Angiogenesis and vasculogenesis are important in embryonic development, inflammation, and wound healing, and also contribute to pathologic conditions such as tumor growth, diabetic retinopathy, rheumatoid arthritis, and chronic inflammatory diseases (see, e.g., USPN 5,318,957; Yancopoulos et a/. (1998) Ce// 93:661-4; Folkman et al. (1996) Ce// 87;1153-5; and Hanahan et al. (1996) Ce// 86:353-64).
  • Both angiogenesis and vasculogenesis involve the proliferation of endothelial cells.
  • Endothelial cells line the walls of blood vessels; capillaries are comprised almost entirely of endothelial cells.
  • the angiogenic process involves not only increased endothelial cell proliferation, but also comprises a cascade of additional events, including protease secretion by endothelial cells, degradation of the basement membrane, migration through the surrounding matrix, proliferation, alignment, differentiation into tube-like structures, and synthesis of a new basement membrane.
  • Vasculogenesis involves recruitment and differentiation of mesenchymal cells into angioblasts, which then differentiate into endothelial cells which then form de novo vessels (see, e.g., Folkman et al. (1996) Ce// 87:1153-5).
  • angiogenesis is involved in the growth of atherosclerotic plaque, diabetic retinopathy, degenerative maculopathy, retrolental fibroplasia, idiopathic pulmonary fibrosis, acute adult respiratory distress syndrome, and asthma. Furthermore, tumor progression is associated with neovascularization, which provides a mechanism by which nutrients are delivered to the progressively growing tumor tissue.
  • the present invention provides methods of reducing angiogenesis in an individual.
  • the methods generally involve administering to the individual an effective amount of an Egfl7 agonist, which agonists may include Eglf7 polypeptides.
  • the methods are useful to treat conditions associated with or resulting from angiogenesis, particularly pathological angiogenesis.
  • the invention further provides methods of treating a condition associated with or resulting from angiogenesis.
  • the present invention features a method of reducing angiogenesis in a mammal.
  • the method generally involves administering to a mammal an Egfl7 agonist in an amount effective to reduce angiogenesis. It is shown herein that Egfl7 acts to inhibit the migration and tube formation by endothelial cells.
  • the present invention also features method of treating a disorder associated with pathological angiogenesis.
  • the invention features a method of inhibiting abnormal fibrovascular growth in a mammal.
  • the abnormal fibrovascular growth is associated with inflammatory arthritis.
  • the invention features a method of inhibiting a proliferative retinopathy in a mammal.
  • the proliferative retinopathy occurs as a result of diabetes in the mammal.
  • the methods generally involve administering to a mammal an Egfl7 agonist in an amount effective to reduce pathological angiogenesis. In some embodiments, the methods further comprise administering a second angiogenesis inhibitor.
  • the present invention further features a method of inhibiting tumor growth in a mammal. In some embodiments, the invention features a method of inhibiting pathological neovascularization associated with a tumor.
  • the methods generally involve administering to a mammal an Egfl7 agonist in an amount effective to reduce angiogenesis associated with a tumor. In some embodiments, the invention further comprises administering an anti-tumor chemotherapeutic agent other than an Egfl7 agonist.
  • Figure 1 Effect of angiogenesis inhibition on tumor growth and survival.
  • Figure 2 Egfl7 structural motifs and expression pattern.
  • Histidine tag was cloned downstream of the CMV promoter in the pHRST-l RES-GFP lentiviral construct (A).
  • a control lentiviral construct with no insert was also created.
  • B Overexpression of Egfl7 in GFP-sorted, lentivirus Egfl7- IRES-GFP transduced T241 cells demonstrated by (C 1 lanes labeled Egfl7) Western blotting of whole-cell extracts with a rabbit polyclonal anti-Egfl7 antibody.
  • T241 tumors show a striking decrease in tumor vasculature in Egfl7 expressing T241 tumors (B) compared to control GFP tumors (A).
  • B Egfl7 expressing T241 tumors
  • A control GFP tumors
  • Cy3-SMA shows colocalization of pericytes and endothelial cells in Egfl7 expressing tumors.
  • Control GFP expressing tumors show staining characteristic of aberrant tumor vasculature with poor association of pericytes with endothelial cells.
  • White arrows indicate striking examples of intimate associations between pericytes (red) and endothelial cells (green) in an Egfl7 expressing tumor.
  • Yellow arrows indicate pericytes detached from tumor vasculature.
  • Egfl7 is a 30 kDa highly conserved endothelial cell-secreted protein containing a signal peptide, two EGF repeats, and a cysteine-rich EM! domain (Fig. 2). Egfl7 is secreted but tightly associated with the extracellular matrix, rendering purification of soluble active protein problematic. During embryonic development, Egfl7 is clearly expressed in a pan-endothelial fashion with strong expression in all vascular beds (Fig. 2), while it is largely absent in resting adult vasculature.
  • treatment means obtaining a desired pharmacologic and/or physiologic effect, e.g., reduction of angiogenesis and/or vasculogenesis.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect.
  • Treatment covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing a disease or condition from occurring in a subject who may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, e.g., arresting its development; or (c) relieving the disease.
  • reduction of angiogenesis and/or vasculogenesis is employed for subject having a disease or condition amenable to treatment by reducing angiogenesis.
  • terapéuticaally effective amount of an Egfl7 agonist is meant an amount of an Egfl7 agonist effective to facilitate a desired therapeutic effect, e.g., a desired reduction of angiogenesis and/or vasculogenesis.
  • a desired therapeutic effect e.g., a desired reduction of angiogenesis and/or vasculogenesis.
  • the precise desired therapeutic effect will vary according to the condition to be treated.
  • Egfl7 agonist is an active Egfl7 polypeptide.
  • EGFL7 peptides which can be used in the methods of the invention, comprise at least about 10 amino acids, usually at least about 12 amino acids, at least about 15 amino acids, and which may include up to or more than 50 amino acids of a EGFL7 peptide, including domains and larger fragments of about 100 amino acids or more; and modifications thereof, and may further include fusion polypeptides as known in the art in addition to the provided sequences. A combination of one or more forms may be used.
  • the EGFL7 sequence may be from any mammalian or avian species, e.g. primate sp., particularly humans; rodents, including mice, rats and hamsters; rabbits; equines, bovines, canines, felines; etc. Of particular interest are the human proteins.
  • VE-statin both of which encode a deduced 273-amino acid protein with a calculated molecular mass of 29.6 kD (see Soncin, et al. (2003) EMBO J. 22: 5700-5711, herein incorporated by reference).
  • the mouse and human EGFL7 proteins both contain an N- terminal cleavable signal peptide followed by 2 epidermal growth factor-like domains, and they share 78% amino acid identity.
  • the mouse Egfl7 is a deduced 278-amino acid protein with a calculated molecular mass of 29 kD.
  • variants of the EGFL7 polypeptide are of interest.
  • Such variants may have substantial sequence similarity to a native EGFL7 sequence, for example SEQ ID NO:1, usually at least about 90% sequence identity; at least about 95% sequence identity; up to at least about 99% sequence identity or more.
  • Such variants may comprise 1, 2, 3, 4, 5, or more amino acid substitutions, deletions or additions, including conservative substitutions.
  • Functional variants may also be assessed by the ability of a variant to activate pathways mediated by the wild-type EGFL7 polypeptide, for example where the variant has an activity at least equal to the wild-type protein; and activity greater than the wild-type protein; or an activity not less than about 25% the activity of the wild-type protein.
  • the activity may be ligand dependent or ligand independent, usually ligand dependent.
  • EGFL7 has been identified as having certain activities, as reported herein, in the inhibition of angiogenesis, and such assays may be performed according to the examples set forth herein to determine activity of an Egfl7 variant.
  • an Egfl7 agonist is meant that the Egfl7 agonist has been separated from components that accompany it in nature.
  • an Egfl7 agonist is substantially pure when it is at least 60%, by weight, free from naturally-occurring organic molecules with which it is naturally associated.
  • the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, Egfl7 agonist.
  • a substantially pure Egfl7 agonist can be obtained, for example, by extraction from a natural source, by recombinant synthesis, by chemically synthesizing the compound, or by a combination of purification and chemical modification. Purity can be measured by any appropriate method, e.g., chromatography, mass spectroscopy, HPLC analysis, etc.
  • An Egfl7 agonist is substantially free of naturally associated components when it is separated from those contaminants which accompany it in its natural state.
  • an Egfl7 agonist which is chemically synthesized or produced in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally associated components.
  • the Egfl7 agonist is generally substantially pure, e.g., at least about 90% pure, at least about 95% pure, or at least about 99% pure.
  • the present invention provides methods of reducing angiogenesis in an individual.
  • the methods generally involve administering to an individual an effective amount of a Egfl7 agonist.
  • the methods are useful to treat conditions and disorders associated with or resulting from angiogenesis, particularly pathological angiogenesis.
  • Egfl7 agonists are useful to treat conditions and disorders associated with and/or resulting from pathological angiogenesis, including, e.g., cancer, atherosclerosis, proliferative retinopathies, excessive fibrovascular proliferation as seen with chronic arthritis, psoriasis, and vascular malformations such as hemangiomas.
  • the present invention provides methods of reducing angiogenesis in an individual.
  • the methods generally involve administering to an individual an effective amount of an Egfl7 agonist.
  • Egfl7 agonists can be identified using readily available methods, including those described in the Example.
  • the ability of a candidate agent to reduce angiogenesis can be assessed in vitro or in vivo using any known method, including, but not limited to, an in vitro Matrigel assay, a disc angiogenesis system, a murine model of hind limb ischemia, a murine model of lung cancer, and the like.
  • the pharmaceutical compositions comprising an Egfl7 agonist described herein can be provided in a wide variety of formulations. More particularly, the Egfl7 agonist can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid (e.g., gel), liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols. [0039] .
  • solid, semi-solid e.g., gel
  • liquid or gaseous forms such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols.
  • Egfl7 agonist formulation used will vary according to the condition or disease to be treated, the route of administration, the amount of Egfl7 agonist to be administered, and other variables that will be readily appreciated by the ordinarily skilled artisan.
  • administration of Egfl7 agonists can be either systemic or local, and can be achieved in various ways, including, but not necessarily limited to, administration by a route that is oral, parenteral, intravenous, intraarterial, inter-pericardial, intramuscular, intraperitoneal, intra-articular, intra-ocular, topical, transdermal, transcutaneous, subdermal, intradermal, intrapulmonary, etc.
  • the Egfl7 agonist may be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds.
  • the following methods and excipients are merely exemplary and are in no way limiting.
  • the Egfl7 agonist can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
  • Formulations suitable for topical, transcutaneous, and transdermal administration may be similarly prepared through use of appropriate suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. Topical formulations may be also utilized with a means to provide continuous administration, for example, incorporation into slow-release pellets or control led-release patches.
  • the Egfl7 agonist can also be formulated in a biocompatible gel, which gel can be applied topically or implanted (e.g., to provide for sustained release of Egfl7 agonist at an internal treatment site).
  • a biocompatible gel which gel can be applied topically or implanted (e.g., to provide for sustained release of Egfl7 agonist at an internal treatment site).
  • Suitable gels and methods for formulating a desired compound for delivery using the gel are well known in the art (see, e.g., US Pat. Nos. 5,801.033; 5,827,937; 5,700,848; and MATRIGELTM).
  • the Egfl7 agonist can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.
  • conventional additives such as lactose, mannitol, corn starch or potato starch
  • binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins
  • disintegrators such as corn starch, potato starch or sodium carboxymethylcellulose
  • lubricants such as talc or magnesium stea
  • the Egfl7 agonist can be utilized in aerosol formulation to be administered via inhalation.
  • the compounds of the present invention can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.
  • the Egfl7 agonist can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases.
  • the compounds of the present invention can be administered rectally via a suppository.
  • the suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.
  • Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more inhibitors.
  • unit dosage forms for injection or intravenous administration may comprise the inhibitors) in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.
  • unit dosage form refers to physically discrete units suitable as unitary dosages for human and/or animal subjects, each unit containing a predetermined quantity of Egfl7 agonist calculated in an amount sufficient to produce the desired reduction in angiogenesis in association with a pharmaceutically acceptable diluent, carrier or vehicle.
  • the specifications for the unit dosage forms of the present invention depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.
  • an Egfl7 agonist is administered in a combination therapy with one or more additional therapeutic agents.
  • exemplary therapeutic agents include therapeutic agents used to treat cancer, atherosclerosis, proliferative retinopathies, chronic arthritis, psoriasis, hemangiomas, etc.
  • the dose of Egfl7 agonist administered to a subject, particularly a human, in the context of the present invention should be sufficient to effect a therapeutic reduction in angiogenesis in the subject over a reasonable time frame.
  • the dose will be determined by, among other considerations, the potency of the particular Egfl7 agonist employed and the condition of the subject, as well as the body weight of the subject to be treated.
  • the size of the dose also will be determined by the existence, nature, and extent of any adverse side- effects that might accompany the administration of a particular compound.
  • the route of administration e.g., the kinetics of the release system (e.g., pill, gel or other matrix), and the potency of the agonist are considered so as to achieve the desired anti-angiogenic effect with minimal adverse side effects.
  • the Egfl7 agonist will typically be administered to the subject being treated for a time period ranging from a day to a few weeks, consistent with the clinical condition of the treated subject.
  • the dosage is adjusted for Egfl7 agonist according to their potency and/or efficacy relative to a standard, e.g., native Egfl7 protein.
  • a dose may be in the range of about 0.01 ⁇ g to 10 mg, given 1 to 20 times daily, and can be up to a total daily dose of about 0.1 ⁇ g to 100 mg. If applied topically, for the purpose of a systemic effect, the patch or cream would be designed to provide for systemic delivery of a dose in the range of about 0.01 ⁇ g to 10 mg.
  • the matrix in which the Egfl7 agonist is administered is designed to provide for a systemic delivery of a dose in the range of about 0.001 ⁇ g to 1 mg. If injected for the purpose of a local effect, the matrix is designed to release locally an amount of Egfl7 agonist in the range of about 0.003 ⁇ g to 1 mg.
  • the dose of Egfl7 agonist can be administered over any appropriate time period, e.g., over the course of 1 to 24 hours, over one to several days, etc. Furthermore, multiple doses can be administered over a selected time period. A suitable dose can be administered in suitable subdoses per day, particularly in a prophylactic regimen. The precise treatment level will be dependent upon the response of the subject being treated. Combination therapy
  • an Egfl7 agonist is administered in a combination therapy with one or more other therapeutic agents, including an inhibitor of angiogenesis; and a cancer chemotherapeutic agent.
  • Suitable chemotherapeutic agents include, but are not limited to, the alkylating agents, e.g. Cisplatin, Cyclophosphamide, Altretamine; the DNA strand-breakage agents, such as Bleomycin; DNA topoisomerase Il inhibitors, including intercalators, such as Amsacrine, Dactinomycin, Daunorubicin, Doxorubicin, Idarubicin, and Mitoxantrone; the nonintercalating topoisomerase Il inhibitors such as, Etoposide and Teniposide; the DNA minor groove binder Plicamycin; alkylating agents, including nitrogen mustards such as Chlorambucil, Cyclophosphamide, Isofamide, Mechlorethamine, Melphalan, Uracil mustard; aziridines such as Thiotepa; methanesulfonate esters such as Busulfan; nitroso ureas, such as Carmustine, Lomustine, Strept
  • the Egfl7 agonist may be administered with other anti-angiogenic agents.
  • Anti-angiogenic agents include, but are not limited to, angiostatic steroids such as heparin derivatives and glucocorticosteroids; thrombospondin; cytokines such as IL-12; fumagillin and synthetic derivatives thereof, such as AGM 12470; interferon- ⁇ ; endostatin; soluble growth factor receptors; neutralizing monoclonal antibodies directed against growth factors such as vascular endothelial growth factor; and the like. Reducing anqio ⁇ enesis in vivo
  • the instant invention provides a method of reducing angiogenesis in a mammal.
  • the method generally involves administering to a mammal an Egfl7 agonist in an amount effective to reduce angiogenesis.
  • An effective amount of an Egfl7 agonist reduces angiogenesis by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or more, when compared to an untreated (e.g., a placebo-treated) control.
  • Whether angiogenesis is reduced can be determined using any known method.
  • Methods of determining an effect of an agent on angiogenesis include, but are not limited to, inhibition of neovascularization into implants impregnated with an angiogenic factor; inhibition of blood vessel growth in the cornea or anterior eye chamber; inhibition of endothelial cell proliferation, migration or tube formation in vitro; the chick chorioallantoic membrane assay; the hamster cheek pouch assay; the polyvinyl alcohol sponge disk assay.
  • Such assays are well known in the art and have been described in numerous publications, including, e.g., Auerbach et al. ((1991) Pharmac. Ther. 51:1-11), and references cited therein.
  • the invention further provides methods for treating a condition or disorder associated with or resulting from pathological angiogenesis.
  • a reduction in angiogenesis effects a reduction in tumor size; and a reduction in tumor metastasis.
  • Whether a reduction in tumor size is achieved can be determined, e.g., by measuring the size of the tumor, using standard imaging techniques.
  • Whether metastasis is reduced can be determined using any known method. Methods to assess the effect of an agent on tumor size are well known, and include imaging techniques such as computerized tomography and magnetic resonance imaging.
  • Any condition or disorder that is associated with or that results from pathological angiogenesis, or that is facilitated by neovascularization is amenable to treatment with an Egfl7 agonist.
  • Conditions and disorders amenable to treatment include, but are not limited to, cancer; atherosclerosis; proliferative retinopathies such as diabetic retinopathy, age- related maculopathy, retrolental fibroplasia; excessive fibrovascular proliferation as seen with chronic arthritis; psoriasis; and vascular malformations such as hemangiomas, and the like.
  • the instant methods are useful in the treatment of both primary and metastatic solid tumors, including carcinomas, sarcomas, leukemias, and lymphomas. Of particular interest is the treatment of tumors occurring at a site of angiogenesis. Thus, the methods are useful in the treatment of any neoplasm, including, but not limited to.
  • the instant methods are also useful for treating solid tumors arising from hematopoietic malignancies such as leukemias (i.e. chloromas, plasmacytomas and the plaques and tumors of mycosis fungoides and cutaneous T-cell lymphoma/leukemia) as well as in the treatment of lymphomas (both Hodgkin's and non-Hodgkin's lymphomas).
  • leukemias i.e. chloromas, plasmacytomas and the plaques and tumors of mycosis fungoides and cutaneous T-cell lymphoma/leukemia
  • lymphomas both Hodgkin's and non-Hodgkin's lymphomas.
  • the instant methods are useful for reducing metastases from the tumors described above either when used alone or in combination with radiotherapy and/or other chemotherape ⁇ tic agents.
  • autoimmune diseases such as rheumatoid, immune and degenerative arthritis
  • various ocular diseases such as diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, retrolental fibroplasia, neovascular glaucoma, rubeosis, retinal neovascularization due to macular degeneration, hypoxia, angiogenesis in the eye associated with infection or surgical intervention, and other abnormal neovascularization conditions of the eye
  • skin diseases such as psoriasis
  • blood vessel diseases such as hemangiomas, and capillary proliferation within atherosclerotic plaques
  • Osier-Webber Syndrome plaque neovascularization
  • telangiectasia hemophiliac joints
  • excessive wound granulation keloids
  • an agent that agonizes Egfl7 -regulated gene is a small molecule, e.g., a small organic or inorganic compound having a molecular weight of more than about 50 daltons and less than about 20,000 daltons, e.g., from about 50 daltons to about 100 daltons, from about 100 daltons to about 200 daltons, from about 200 daltons to about 500 daltons, from about 500 daltons to about 1000 daltons, from about 1000 daltons to about 2500 daltons, from about 2500 daltons to about 5000 daltons, from about 5000 daltons to about 7,500 daltons, from about 7,500 daltons to about 10,000 daltons, from about 10,000 daltons to about 15,000 daltons, or from about 15,000 daltons to about 20,000 daltons.
  • a small organic or inorganic compound having a molecular weight of more than about 50 daltons and less than about 20,000 daltons, e.g., from about 50
  • Agents may comprise functional groups necessary for structural interaction with proteins and/or nucleic acids, e.g., hydrogen bonding, and may include at least an amine, carbonyl, hydroxy! or carboxyl group, and may contain at least two of the functional chemical groups.
  • the agents may comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • Agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.
  • an agent that agonizes Egfl7 gene is a nucleic acid, e.g., an antisense RNA, an interfering RNA (including short interfering RNA; "siRNA”), a ribozyme, and the like, usually a nucleic acid encoding an Egfl7 polypeptide, operably linked to a promoter active in the cells of interest.
  • An Egfl7 encoding polynucleotide sequence e.g., cDNA or genomic DNA
  • the vector components generally include, but are not limited to, one or more of the following: an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.
  • Egfl7 polypeptides may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, e.g. a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide.
  • a heterologous polypeptide e.g. a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide.
  • the signal sequence may be a component of the vector, or it may be a part of the Egfl7 polypeptide DNA that is inserted into the vector.
  • the heterologous signal sequence selected preferably is one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell.
  • the native signal sequence may be used, or other mammalian signal sequences may be suitable, such as signal sequences from other animal Egfl7 polypeptide, and signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders, for example, the herpes simplex gD signal.
  • Expression vectors will contain a promoter that is recognized by the host organism and is operably linked to the Egfl7 coding sequence. Promoters are untranslated sequences located upstream (5') to the start codon of a structural gene (generally within about 100 to 1000 bp) that control the transcription and translation of particular nucleic acid sequence to which they are operably linked. Such promoters typically fall into two classes, inducible and constitutive. Inducible promoters are promoters that initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, e.g., the presence or absence of a nutrient or a change in temperature. A large number of promoters recognized by a variety of potential host cells are well known.
  • Both a native Egfl7 polypeptide promoter sequence and many heterologous promoters may be used to direct expression of a Egfl7 polypeptide.
  • heterologous promoters are preferred, as they generally permit greater transcription and higher yields.
  • Transcription from vectors in mammalian host cells may be controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and most preferably Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter, PGK (phosphoglycerate kinase), or an immunoglobulin promoter, from heat-shock promoters, provided such promoters are compatible with the host cell systems.
  • viruses such
  • the early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication.
  • the immediate early promoter of the human cytomegalovirus is conveniently obtained as a Hindlll E restriction fragment.
  • Transcription by higher eukaryotes is often increased by inserting an enhancer sequence into the vector.
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, which act on a promoter to increase its transcription. Enhancers are relatively orientation and position independent, having been found 5' and 3' to the transcription unit, within an intron, as well as within the coding sequence itself.
  • enhancer sequences are now known from mammalian genes (globin, elastase, albumin, ⁇ - fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. The enhancer may be spliced into the expression vector at a position 5' or 3' to the coding sequence, but is preferably located at a site 5' from the promoter.
  • Plasmids from the transformants are prepared, analyzed by restriction endonuclease digestion, and/or sequenced.
  • transient expression involves the use of an expression vector that is able to replicate efficiently in a host cell, such that the host cell accumulates many copies of the expression vector and, in turn, synthesizes high levels of a desired polypeptide encoded by the expression vector.
  • Transient expression systems comprising a suitable expression vector and a host cell, allow for the convenient positive identification of polypeptides encoded by cloned DNAs, as well as for the rapid screening of such polypeptides for desired biological or physiological properties.
  • Egfl7 polypeptide in preclinical models may utilize an adenovirus technology (Ad TRE (Tetracycline response element)) which permits rigorously controlled expression of adenovirally carried transgenes under the control of the tetracycline operator/promoter system.
  • Ad TRE Tetracycline response element
  • Expression of the desired transgene engineered in the Ad TRE adenovirus utilizes co-infection with Ad tTA (tetracycline transactivator), which is an adenovirus which expresses the requisite transcription factor for the TRE system.
  • a stimulator of therapeutic angiogenesis is administered to an individual in need thereof.
  • the stimulator of angiogenesis is an active agent that inhibits Egfl7 activity or expression, and increases angiogenesis.
  • the instant invention provides a method of increasing or stimulating angiogenesis in a mammal. The method generally involves administering to a mammal an active agent in an amount effective to inhibit Egfl7 activity, thereby increasing angiogenesis.
  • An effective amount of an Egfl7 inhibitor increases angiogenesis by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 2- fold, at least about 5-fold, at least about 10-fold, or more, when compared to an untreated (e.g., a placebo-treated) control.
  • Stimulation of angiogenesis is useful to treat a variety of conditions that would benefit from stimulation of angiogenesis, stimulation of vasculogenesis, increased blood flow, and/or increased vascularity.
  • Examples of conditions and diseases amenable to treatment according to the method of the invention related to increasing angiogenesis include any condition associated with an obstruction of a blood vessel, e.g., obstruction of an artery, vein, or of a capillary system.
  • Specific examples of such conditions or disease include, but are not necessarily limited to, coronary occlusive disease, carotid occlusive disease, arterial occlusive disease, peripheral arterial disease, atherosclerosis, myointimal hyperplasia (e.g., due to vascular surgery or balloon angioplasty or vascular stenting), thromboangiitis obliterans, thrombotic disorders, vasculitis, and the like.
  • Examples of conditions or diseases that can be prevented using the methods of the invention include, but are not necessarily limited to, heart attack (myocardial infarction) or other vascular death, stroke, death or loss of limbs associated with decreased blood flow, and the like. Screening Assays
  • the present invention further provides methods of identifying an agent that modulates angiogenesis.
  • the methods may involve contacting a cell that is responsive to Egfl7 with a test agent, for example in a competitive assay with Egfl7; and assessing the effect of the test agent upon Egfl7 mediated effects.
  • a test agent for example in a competitive assay with Egfl7
  • an agonist or antagonist of Egfl7 may be designed to bind to or mimic the activity of Egfl7 F e.g. in the design of a polypeptide or peptidomimetic agent.
  • Such an agent may then be tested in any standard angiogenesis assay to confirm activity.
  • agent encompass numerous biological and chemical classes, including synthetic, semi-synthetic, or naturally-occurring inorganic or organic molecules, including synthetic, recombinant or naturally-occurring polypeptides and nucleic acids (e.g., nucleic acids encoding a gene product, antisense RNA, siRNA, and the like).
  • candidate agents or “test agents” particularly include those found in large libraries of synthetic or natural compounds.
  • agents of interest include small organic or inorganic compounds having a molecular weight of more than 50 and less than about 2,500 daltons.
  • Candidate agents may comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and may include at least an amine, carbonyl, hydroxyl or carboxyl group, and may contain at least two of the functional chemical groups.
  • the agents may comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • Agents, particularly candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.
  • a variety of other reagents may be included in the screening assay. These include reagents like salts, neutral proteins, e.g. albumin, detergents, etc that are used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Reagents that improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc. may be used.
  • the components of the assay mixture are added in any order that provides for the requisite binding or other activity. Incubations are performed at any suitable temperature, typically between 4°C and 40 0 C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high-throughput screening. Typically between 0.1 and 1 hour will be sufficient.
  • Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pi, picoliter(s); s or sec, second(s); mi ⁇ , minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscularly); i.p., intraperitoneal(ly); s.c, subcutaneous(ly); and the like.
  • Anti-angiogenic therapy targeting not the tumor cells themselves but rather tumor blood vessels has demonstrated great promise in human cancer patients, most notably in a randomized phase III trial of the anti-VEGF monoclonal antibody Avastin in metastatic colorectal cancer.
  • VEGF blockade in animal models and humans, such treatment has been nevertheless characterized by progressive disease and largely modest survival improvements. Accordingly, we have exploited our adenoviral approach to greatly facilitate the search for novel anti-angiogenic compounds which could be either superior to VEGF blockade, or have additive/synergistic effects when combined with VEGF blockade.
  • Eglf7 is a recently described secreted protein containing two EGF repeats which is expressed in a pan-endothelial fashion during mouse development; however, its potential anti-angiogenic properties in any tumor type, have not been examined.
  • Egfl7 also known as VE-Statin
  • VE-Statin the secreted peptide
  • Egfl7 is a 30 kDa highly conserved endothelial cell- secreted protein containing a signal peptide, a cysteine-rich EMI domain and two EGF repeats.
  • B During development, Egfl7 is expressed in a panendothelial fashion with strong expression in all vascular beds.
  • CD31 staining of whole mount preparations of murine ear three days after subcutaneous injection with adenovirus show the normal vascular patterning of the reticular dermis of the ear after treatment with control virus (A).
  • Ad-VEGF induces robust cellular changes in the structural features of the blood vessels and increases vascularity (B).
  • B increases vascularity
  • C co-injection with Ad-Egfl7 results in the inhibition of these VEGF-induced changes in the vasculature
  • Egfl7 Lentiviral constructs expressing Egfl7-I RES-G FP or IRES-GFP alone were created by cloning the Egfl7 cDNA tagged with 6X Histidine into the pHRST-l RES-GFP backbone. Lentiviruses were packaged in 293T cells by triple transfection with Lipofectamine 2000 (Invitrogen). T241 fibrosarcoma cells were infected with Egfl7 expressing viruses and GFP expressing, control viruses in minimal volume in the presence of 1X Polybrene.
  • Transduced T241 cells were isolated by Fluorescence Activated Cell Sorting (FACS) at the Stanford University FACS core as GFP positive cells and plated at a density of 1 cell/100 ⁇ l_ in individual wells of a 96 well plates. Individual GFP positive clones were expanded and expression of the Egfl7 gene was confirmed by Western Blotting with a rabbit polyclonal anti-Egfl7 antibody.
  • FACS Fluorescence Activated Cell Sorting
  • tumor cells were plated at a density of 500 cells/well on five 24 well plates. One plate was harvested every 24 hours and stored at -80 0 C. All 5 plates were subsequently stained with 200 ⁇ L/well of CyQuant GR dye (excitation/emission maxima-480/530, Molecular Probes) and read in a Flexstation Il 384 fluorescence microplate reader. The fluorescence unit of each well is directly proportional to the number of cells and represents the mean of 3 separate wells.
  • mice having tumors around 100 cubic mm in volume were taken from each group to be perfused and analyzed by immunofluorescence. After anesthetization with 1X Avertin, mice were perfused systemically with 1% paraformaldehyde through the left ventricle for 2 minutes at a pressure of 120 mmHg. Fixed tumors were cryoem bedded and sectioned to a thickness of 10 ⁇ m and stored at -80 0 C. Before antibody labeling, sectioned tumors were thawed at room temperature, permeabilized with 0.3% Triton X-100 in 1X PBS (TPBS) and blocked with 5% goat serum in TPBS.
  • TPBS Triton X-100 in 1X PBS
  • rat monoclonal anti-mouse CD31 PECAM-1 , clone MEC 13.3, 1 :250 in 5% goat serum in TPBS, Pharmingen, San Diego, CA.
  • the samples were washed with 1X PBS and then labeled with FITC conjugated goat anti-rat IgG (1 :400 in 5% goat serum in TPBS, Jackson ImmunoResearch, West Grove, PA) for 4 hours in the dark. Unbound secondary antibodies were washed off by 1 X PBS and samples were then fixed with 4% Paraformaldehyde and mounted for subsequent immunofluorescent visualization.
  • Endothelial Cells were seeded for lentiviral infection at a density of 2.5 x 10 5 cells/well of a 6 well plate in 3 ml. of EBM2-EGM2 media (Cambrex).
  • EBM2-EGM2 media Cell-derived Endothelial Cells
  • a separate well was also infected with 300 ⁇ L unconcentrated Lentivirus expressing only GFP in 300 ⁇ L EBM2-EGM2 media with 0.6 ⁇ L 1000X Polybrene.
  • Egfl7 Expressed by Lentiviral Transduced T241 Fibrosarcoma Cells.
  • T241 fibrosarcoma cells transduced with lentiviruses encoding Egfl7 and control GFP were FACS sorted and expanded.
  • Two T241 clones were isolated and shown to stably express Egfl7 in comparison to control cells transduced with a GFP lentivirus (Fig 5C).
  • Fig 5C A comparison of the proliferation of Egfl7 expressing T241 cells to control GFP expressing cells reveals that Egfl7 expression does not adversely affect tumor cell proliferation (Fig. 6B).
  • Size matched tumors from both Egfl7 expressing and GFP expressing tumors were harvested from perfused mice, sectioned, and stained with anti-CD31 FITC conjugated antibodies to visualize the vasculature.
  • Tumor sections taken from control GFP expressing tumors show abundant CD31 staining (Fig. 7A) while sections from Egfl7 tumors have little to no positive CD31 staining (Fig. 7B).
  • Pericytes or perivascular cells, are multipotent support cells found in close association with endothelial cells of the microvasculature.
  • the close spatial arrangement of perictyes and endothelial cells allows for communication between these cell types in the form of soluble factors and cell-cell contacts.
  • the presence of closely attached pericytes is a hallmark of mature blood vessels.
  • blood vessels formed through VEGF induced tumor angiogenesis are often characterized by a loose association or a complete absence of pericytes. This abnormality has been speculated to contribute to excessive vessel permeability caused by pathological VEGF activity.
  • Pericytes visualized with a Cy3 conjugated anti-Smooth Muscle Actin antibody, in GFP expressing T241 subcutaneous tumors are detached from neighboring capillaries.
  • pericytes in Egfl7 expressing tumors colocalize closely with FITC- CD31 labeled endothelial cells, thereby implying that the vasculature in these tumors is more normalized.
  • Normalization of tumor vasculature exemplified by improved vessel integrity and the recruitment of supporting perivascular cells, is a general sign of VEGF blockade.
  • EgfI7 inhibits VEGF in an autocrine manner through inhibiting endothelial cell proliferation, migration, and/or tube formation.
  • Egfl7 lentivirus or GFP lentivirus were first transduced with Egfl7 lentivirus or GFP lentivirus to stimulate overexpression of Egfl7 as shown by Western blotting with a rabbit monoclonal anti-Egfl7 antibody (Fig. 6A).
  • HUVECs that express the inserted gene could also be followed as GFP positive cells (Fig.
  • the angiogenic switch is a vital step in the development of macroscopic and metastatic tumors and is initiated by tumor secreted angiogenic activators, such as VEGF, that stimulate the proliferation, migration, and differentiation of neighboring endothelial cells.
  • Egfl7 is an endothelial cell secreted protein. Due to the difficulty of obtaining significant amounts of purified Egfl7 protein, 6X Histidine tagged Egfl7 was overexpressed in T241 fibrosarcoma cells and implanted into adult mice.
  • Egfl7 is synthesized and posttranslationally modified within mammalian cells and is present in the local environment of the tumor mass where there is intimate contact between tumor cells and vascular endothelial cells.
  • Expression of Egfl7 in this manner led to a striking decrease in vascularity and a potent inhibition of tumor growth in vivo, suggesting that Egfl7 restrains tumor growth by inhibiting VEGF induced tumor angiogenesis.
  • Vascular endothelial cells respond to angiogenic stimuli, in the form of VEGF or other soluble activators, by proliferating, migrating towards the source of chemoattractant, and differentiating into nascent vasculature.
  • Egfl7 the antiangiogenic activity of Egfl7 in tumors could be due to attenuation of one or more of these three cellular responses.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Marine Sciences & Fisheries (AREA)
  • Zoology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne des procédés pour moduler l'angiogenèse dans un individu, les procédés mettant en jeu d'une manière générale l'administration à un individu d'un agent qui module l'expression ou l'activité de Egf17, où un agoniste de Egf17, comprenant un polypeptide Egf17, diminue l'angiogenèse. Dans un mode de réalisation, les procédés de l'invention concernent l'inhibition d'une angiogenèse pathologique par l'augmentation de l'activité de Egf17, lequel procédé peut être réalisé conjointement avec l'administration d'un ou plusieurs autres agents anti-angiogéniques.
PCT/US2007/016127 2006-07-17 2007-07-16 Procédés de modulation de l'angiogenèse WO2008010986A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US83151506P 2006-07-17 2006-07-17
US60/831,515 2006-07-17

Publications (2)

Publication Number Publication Date
WO2008010986A2 true WO2008010986A2 (fr) 2008-01-24
WO2008010986A3 WO2008010986A3 (fr) 2008-03-13

Family

ID=38957304

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2007/016127 WO2008010986A2 (fr) 2006-07-17 2007-07-16 Procédés de modulation de l'angiogenèse

Country Status (2)

Country Link
US (1) US7947278B2 (fr)
WO (1) WO2008010986A2 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010034155A1 (fr) * 2008-09-25 2010-04-01 中南大学湘雅医院 Kit de détection d’egfl7 dans des échantillons pour le diagnostic du carcinome hépatocellulaire et son application
EP2831093A4 (fr) * 2012-03-27 2015-11-04 London Health Sci Ct Res Inc Polypeptides de liaison et/ou de ciblage d'egfl 7 et procédés pour l'inhibition de l'angiogenèse

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013061112A1 (fr) 2011-10-24 2013-05-02 Centre National De La Recherche Scientifique Utilisation de modulateurs de egfl7 afin de favoriser ou d'inhiber la migration de cellules immunitaires à travers l'endothélium vasculaire
JP6116561B2 (ja) * 2012-06-15 2017-04-19 オリンパス株式会社 画像処理装置、顕微鏡システム及び画像処理方法
US20140065112A1 (en) * 2012-08-24 2014-03-06 Texas Heart Institute Compositions and methods for mesenchymal/stromal stem cell rejuvenation and tissue repair by enhanced co-expression of telomerase and myocardin

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5318957A (en) * 1992-09-02 1994-06-07 The United States Of America As Represented By The Department Of Health And Human Services Method of stimulating angiogenesis
EP1196561A2 (fr) 1999-07-02 2002-04-17 Sagami Chemical Research Center Proteines humaines ayant des domaines hydrophobes et adn codant pour ces proteines
CA2454937A1 (fr) * 2001-07-25 2003-02-13 Lorantis Limited Modulateurs de signalisation de notch utilises en immunotherapie
FR2851249A1 (fr) 2003-02-17 2004-08-20 Commissariat Energie Atomique Nouveau facteur soluble secrete par les cellules endotheliales des vaisseaux sanguins, capable d'inhiber le recrutement des cellules perivasculaires du type musculaire lisse et ses applications
AU2005249377A1 (en) * 2004-04-14 2005-12-15 Genentech, Inc. Compositions and methods comprising an EGFL7 antagonist for modulating vascular development

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010034155A1 (fr) * 2008-09-25 2010-04-01 中南大学湘雅医院 Kit de détection d’egfl7 dans des échantillons pour le diagnostic du carcinome hépatocellulaire et son application
EP2831093A4 (fr) * 2012-03-27 2015-11-04 London Health Sci Ct Res Inc Polypeptides de liaison et/ou de ciblage d'egfl 7 et procédés pour l'inhibition de l'angiogenèse
US9789213B2 (en) 2012-03-27 2017-10-17 London Health Sciences Centre Research Inc. EGFL7 targeting and/or binding polypeptides and methods for inhibiting angiogenesis

Also Published As

Publication number Publication date
WO2008010986A3 (fr) 2008-03-13
US20080113911A1 (en) 2008-05-15
US7947278B2 (en) 2011-05-24

Similar Documents

Publication Publication Date Title
AU2008289462B2 (en) Compositions of humanized notch fusion proteins and methods of treatment
EP1259248B1 (fr) Procedes de traitement de cancers exprimant le facteur d de croissance endotheliale vasculaire
KR20130004568A (ko) 항-전이성 요법에서 ａｘｌ 신호전달의 저해
JP2010536855A5 (fr)
US20090036369A1 (en) Anti-tumor agents comprising r-spondins
KR20220062677A (ko) 신데칸 2의 의학적 용도
JP2015178507A (ja) Ｎｏｔｃｈ３シグナル伝達のデコイインヒビターとしてのヒトｎｏｔｃｈ３に基づく融合タンパク質
JP2010068806A (ja) Ｖｅｇｆ−関連タンパク質
Besschetnova et al. Regulatory mechanisms of anthrax toxin receptor 1-dependent vascular and connective tissue homeostasis
US20210196795A1 (en) Therapeutic use of vegf-c and ccbe1
US7947278B2 (en) Methods of modulating angiogenesis
DK2318440T3 (en) ANTIBODIES THAT BIND TO adrenomedullin RECEPTORS AND USES THEREOF AS A PHARMACEUTICAL
EP1900374B1 (fr) Agent angiogénétique contenant de l adrénoméduline comme principe actif
MX2014005318A (es) Terapia genica para la neuropatia diabetica usando una isoforma hgf.
US20100278810A1 (en) Methods of Modulating Angiogenesis
KR20150140686A (ko) 신증후군 및 관련 병태의 치료 방법
EP1519193B1 (fr) Procédés de détection de cancers exprimant le facteur D de croissance endothéliale vasculaire
JP2004506595A (ja) 血管形成及び心臓血管新生の促進又は阻害
JP2013253079A (ja) 生物学的に活性なヒト尿中トリプシンインヒビターのＦｃ融合タンパク質並びにその調製および使用
TWI723247B (zh) 重組蛋白及其用途
US7622443B2 (en) Method for inhibiting pro-angiogenic activities of endothelial cells selectively at a site of neoangiogenesis in a mammal by administration of the extracellular domain of D1-1 polypeptides
Skuli et al. Endothelial HIF-2α regulates murine pathological angiogenesis and revascularization processes.
Sarkar et al. Angiogenesis, Its Importance, and Implication in Tumor Growth and Progression and in Anticancer Therapy: An Update
JP2024067021A (ja) オンコスタチンｍ受容体シグナリング制御による非アルコール性脂肪性肝疾患、肝硬変又は肝癌の予防と治療
KR20140032407A (ko) 케모카인 발현 조절제

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 07836088

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

NENP Non-entry into the national phase

Ref country code: RU

122 Ep: pct application non-entry in european phase

Ref document number: 07836088

Country of ref document: EP

Kind code of ref document: A2

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载